This invention relates generally to ultrathin polymeric membranes. More specifically, the invention relates to polyelectrolyte multilayer films on substrates, and the variations of surface properties of such films to control sorption of charged molecules into or onto such films.
Recently, ultrathin polymeric membranes have been prepared using charged polymers, or polyelectrolytes, which are alternately deposited on a substrate or substratum. See Decher and Schlenoff, Eds., Multilayer Thin Films—Sequential Assembly of Nanocomposite Materials, Wiley-VCH, Weinheim (2003); Decher, Science 277, 1232 (1997); and Decher, Hong, and Schmitt, Thin Solid Films 210/211, 831 (1992). For example, a buildup of multilayers may be accomplished by alternating dipping, i.e., cycling a substrate between two reservoirs containing aqueous solutions of polyelectrolytes of opposite charge, with an optional rinse step in polymer-free solution following each immersion. Each cycle adds a layer of polymer via electrostatic forces to the oppositely-charged surface and reverses the surface charge thereby priming the film for the addition of the next layer. Films prepared in this manner tend to be uniform, follow the contours and irregularities of the substrate, and are typically between about 10 nm and about 10,000 nm thick. The thickness of a film depends on many factors, including the number of layers deposited, the ionic strength of the solutions, the types of polymers, the deposition time, the solution pH, the temperature, and the solvent used. Although studies have shown that the substantial interpenetration of the individual polymer layers results in little composition variation over the thickness of a film, such polymer thin films are, nevertheless, referred to as polyelectrolyte multilayers (PEMUs). Though recently developed, PEMUs are being used in a wide variety of fields including light emitting devices, nonlinear optics, sensors, enzyme active thin films, electrochromics, conductive coatings, patterning, anticorrosion coatings, antistatic coatings, lubricating films, biocompatibilization, dialysis, and as selective membranes for the separation of gaseous and dissolved ionic species. See Fou et al., J. Appl. Phys. 79, 7501 (1996); Decher et al., J. Biosens. Bioelect. 9, 677 (1994); Sun et al., Macromol. Chem. Phys. 197, 147 (1996); Onda et al., Biotech Bioeng 51, 163 (1996); Lvov et al., J. Am. Chem. Soc. 120, 40733 (1998); Laurent et al., Langmuir 13, 1552 (1997); Stepp et al., J. Electrochem. Soc. 144, L155 (1997); Cheung et al., Thin Solid Films 244, 985 (1994); Hammond et al., Macromolecules 28, 7569 (1995); Huck et al., Langmuir 15, 6862 (1999); Stroeve et al., Thin Solid Films 284, 708 (1996); Levasalmi et al., Macromolecules 30, 1752 (1997); Harris et al., Langmuir 16, 2006 (2000); Krasemann et al., 16, 287 (2000); Harris et al., J. Am. Chem. Soc. 121, 1978 (1999); Harris et al., Chem. Mater. 12, 1941 (2000). In fact, PEMUs are particularly suited for use as selective membranes because they are uniform, rugged, easily prepared on a variety of substrates, continuous, resistant to protein adsorption, have reproducible thicknesses, may be made very thin to allow high permeation rates, and may be made from a wide range of compositions. The resulting PEMU systems may be used in applications employing charge reversal for hosting/releasing natural and synthetic compositions such as polypeptides (e.g., proteins), dyes, pharmaceuticals, and the like. See, e.g., Matthews, J. R.; Tuncel, D.; Jacobs, R. M.; Bain, C. D.; Anderson, H. L. J. Am. Chem. Soc. 2002, 125, 6428-6433. See also Harper et al. (PCT Application Publication Number WO 03/101494 A1), which discloses polyion films that, on switching their charge by changing solution pH, also release charged molecules. In trapping and release of charged molecules, it is generally desired that the efficiency of release be maximized, that is, as much of the trapped material as possible should be released in the release step.
Biofunctional thin films with controlled bulk or surface properties have been made by the alternating adsorption of synthetic and/or natural macromolecules to various substrates. (See Decher, G., Schlenoff, J. B. Multilayer Thin Films—Sequential Assembly of Nanocomposite Materials; Wiley-VCH: Weinheim, Germany, 2003.) The wide variety of charged nanocomponents incorporated into these “polyelectrolyte multilayers” (PEMUs) includes biomolecules such as DNA, immunoglobulin, glucose oxidase, and cytochrome c. These biomolecular thin films offer applications as biosensors, nano-filtration, bioreactors, and protein capsules. See Lvov, Y., Möhwald, H. Protein Architecture: Interfacial Molecular Assembly and Immobilization Biotechnology; Marcel Dekker: New York, 2000, Pei, R. J.; Cui, X. Q.; Yang, X. R.; Wang, E. K. Biomacromolecules 2001, 2, 463-468, Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir 1997, 13, 3427-3433, Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117-6123, Ichinose, I.; Takaki, R.; Kuroiwa, K.; Kunitake, T. Langmuir 2003, 19, 3883-3888, Lahav, M.; Kharitonov, A. B.; Katz, O.; Kunitake, T.; Willner, I. Anal. Chem. 2001, 73, 720-723, Tieke, B.; van Ackern, F.; Krasemann, L.; Toutianoush, A. Eur. Phys. J. E. 2001, 5, 29-39, Onda, M.; Lvov, Y.; Ariga, K.; Kunitake, T. Biotechnol. Bioeng. 1996, 51, 163-167, and Lvov, Y.; Caruso, F. Anal. Chem. 2001, 73, 4212-4217. Although multiple “electrostatic” interactions between synthetic and natural charged polymers might be presumed to modify protein conformations, individual interactions are actually quite weak, and, under certain conditions, proteins embedded into PEMUs during the buildup process maintain a secondary structure close to their native form, which renders such PEMUs bioactive. See Dubas, S. T.; Schlenoff, J. B. Langmuir 2001, 17, 7725-7727 and Schwinte, P.; Voegel, J. C.; Picart, C.; Haikel, Y.; Schaaf, P.; Szalontai, B. J. Phys. Chem. B 2001, 105, 11906-11916.
A recent area of interest addresses protein interactions with multilayer-coated surfaces. Fine tuning of protein adsorption at the solid/liquid interface is critical in certain areas of materials science and biomedical engineering. See Horbett, T. A. Acs Sym. Ser. 1995, 602, 1-23. Systems for delivery or biosensors, for example, bear modified surfaces designed to enhance or minimize protein adsorption. See Elbert, D. L.; Hubbell, J. A. Annu. Rev. Mater. Sci. 1996, 26, 365-394 and Szleifer, I. Curr. Opin. Solid St. M. 1997, 2, 337-344. The latter goal is generally desirable for blood-contacting devices, chromatographic supports, contact lenses, and immunoassays, to name a few. Due to their ease of use and water compatibility, PEMUs have been investigated as surface-modifying agents for protein interactions. See Muller, M.; Rieser, T.; Köthe, M.; Kessler, B.; Brissova, M.; Lunkwitz, K. Macromol. Symp. 1999, 145, 149-159, Müller, M.; Brissova, M.; Rieser, T.; Powers, A. C.; Lunkwitz, K. Materials Science & Engineering C-Biomimetic and Supramolecular Systems 1999, 8-9, 163-169, Müller, M.; Rieser, T.; Lunkwitz, K.; Meier-Haack, J. Macromolecular Rapid Communications 1999, 20, 607-611, Ladam, G.; Gergely, C.; Senger, B.; Decher, G.; Voegel, J. C.; Schaaf, P.; Cuisinier, F. J. G. Biomacromolecules 2000, 1, 674-687, (21) Ladam, G.; Schaaf, P.; Cuisinier, F. J. G.; Decher, G.; Voegel, J. C. Langmuir 2001, 17, 878-882, Müller, M.; Rieser, T.; Dubin, P. L.; Lunkwitz, K. Macromolecular Rapid Communications 2001, 22, 390-395. Since protein adsorption triggers further cellular or tissue responses, it is important to study the mechanism by which proteins adsorb onto these thin films. Interactions of more biocomplex systems, such as cells, with PEMUs, a topic of more recent interest, were studied, and different designs for cytophilic/cytophobic PEMUs has been proposed. See Richert, L.; Lavalle, P.; Vautier, D.; Senger, B.; Stoltz, J. F.; Schaaf, P.; Voegel, J. C.; Picart, C. Biomacromolecules 2002, 3, 1170-1178 and Mendelsohn, J. D.; Yang, S. Y.; Hiller, J.; Hochbaum, A. I.; Rubner, M. F. Biomacromolecules 2003, 4, 96-106.
Separation and purification of proteins is often accomplished by ionic exchange chromatography (IEC), which relies on electrostatic interactions between proteins and charged groups on a stationary phase or support. IEC is the most popular chromatographic technique in protein purification, accounting for about 40% of the chromatographic steps included in purification protocols. See Wheelwright, S. M. Protein Purification: Design and Scale-Up of Downstream Processing; Hanser: Munich, Germany, 1991 and Choudhary, G.; Horvath, C. Ion exchange chromatography. Methods Enzymol. 1996, 43, 47-82. Ion exchange, also known as ion displacement, chromatography, offers high resolving power, and high protein-binding capacity.
Ion exchange chromatography typically employs a column (column chromatography) or a plate (thin layer chromatography) filled or coated, respectively, with material having affinity for the protein. A solution containing the protein is passed through the column or imbibed into one edge of the thin later, respectively. In batch separation/purification mode, proteins adhere to the stationary phase, and those species having lower affinity for the stationary phase do not. Immobilized proteins are then released from the column using a chemical or physical stimulus. In column chromatographic purification mode, a solution termed the “mobile” phase is passed continuously though the column and a complex mixture comprising two or more components to be separated is introduced at the head of the column. The components exhibit different degrees of interaction with the stationary phase, such that components travel at different speeds down the column. In a successful separation, components elute at different times from the column and may be detected for the purposes of qualitative and quantitative analysis, or, where purification is the major goal, may be collected for further use. If the stationary phase is present as a thin layer, usually supported on a plate, mixtures are introduced to one edge of the layer and mobile phase is imbibed continually into this edge, transporting components up the layer.
Column batch mode is advantageous because of its simplicity and the fact that loadings, indicated by gram of protein per gram of stationary phase, of material to be purified are greater than in column chromatographic mode. Thus, if large quantities of purified material are desired using column chromatography, many column chromatographic runs must be performed on small aliquots, or extremely large diameter columns must be employed, to obtain protein in quantity. On the other hand, column chromatographic mode offers better resolution between multiple components. In the pharmaceutical industry, commercially important materials, including large and small molecules, such as drugs, are often separated chromatographically by large columns. It is economically viable to do so because the product commands a very high premium.
In column batch mode, and in chromatographic mode, the material with which protein interacts is frequently coated on particulate support that is then packed into the column. Alternatively, the column is filled with a polymer or an inorganic mixture which may be turned into a solid, porous material, which is known as a “monolithic” column. See Zou et al., Monolithic stationary phased for liquid chromatography and capillary electrochromatography, Journal of Chromatography A, 954, 5-32 (2002). The porous monolithic column may be coated with stationary phase.
In its simplest form, a batch separation may be performed by mixing the stationary phase with the mixture, allowing the components to be separated/purified to adhere, rinsing the undesired, non-adhering material away, then releasing the components of interest from the stationary phase. Column batch separation is advantageous because of the convenience of manipulating the solutions used for processing.
In some instances, a protein in solution may be considered pure but too dilute. Such a situation may arise during the course of protein isolation or purification using a series of steps including, but not limited to, dialysis through a semipermeable membrane. While it is possible to reduce the volume of solvent by evaporation, including “freeze drying,” this may denature the protein. Adsorbing the protein from a certain volume of solution onto a stationary phase, then releasing it into a smaller volume is an effective way of concentrating or purifying it.
In batch separation mode, in order to release protein adsorbed to a stationary phase, a stimulus must be added that decreases the interaction of the protein with the stationary phase. Since the interaction between protein and stationary phase is often electrostatic, or charge-pairing, an effective way to remove protein from a stationary phase is to add ionic material, or “salt,” which competes with the protein for the charged sites on the stationary phase. If sufficient salt is added, it occupies the charged adsorption sites on the stationary phase and protein is released. This mechanism may be thought of as ion exchange between salt ions and charged protein units, although the mechanism may also be explained in terms of electrostatic screening of attractive interactions between protein and stationary phase. A major disadvantage of the ion exchange mechanism for protein release is that it often requires large amounts of salt, which end up mixed with protein, to accomplish this. The salt may be removed with a dialysis step, but this adds additional time and inconvenience to the purification procedure. In addition, some proteins may be unstable in the presence of the concentration or type of salt required to release them from the stationary phase.
Alternatively, the interaction between protein and stationary phase may be controlled by the pH of the solution. The sign and magnitude of the charge on proteins depends on the solution pH, as the charge on proteins is carried by weak acids, such as carboxylates (—COOH), or weak bases, such as primary, secondary, and tertiary amines. Thus, at high pH (basic conditions) amines are deprotonated and neutral, and carboxylate groups are deprotonated and charged. For proteins, there is a pH at which there are equal numbers of positive and negative charges on the biomolecule, and it is thus electrically neutral. This is termed the isoelectric point, or pI. At pH above the isoelectric point, the protein has a net negative charge and at pH below pI, proteins bear a net positive charge. Proteins that tend to have a preponderance of positive charge at physiological pH are often termed “basic” proteins.
Assuming the stationary phase is relatively insensitive to pH, if interactions between oppositely-charged stationary phase and protein are largely electrostatic, it is reasoned that removal of a net charge on the protein will reduce or remove this interaction, and further change in pH will actually cause the protein to adopt the same charge as the stationary phase, leading to repulsion. Therefore, if pH change is to be used as a protein removal stimulus, a protein may be adsorbed to a stationary phase under pH conditions where the two have the opposite charge and therefore interact strongly. The pH is then changed to a value sufficient to reduce the charge, and therefore charge interaction, on the protein, releasing it from the stationary phase.
In column chromatographic mode it critical to control finely the interaction between species being separated and stationary phase. Often, the nature of the mobile phase passing through the column is changed by including a proportion of organic solvent. Since many biomolecules are potentially denatured, or degraded, in the presence of organic solvent, alternative ways must be found to fine-tune the interactions with the stationary phase. The addition of salt, or control of ionization of biomolecule or stationary phase by pH, again, offers methods for precise control of protein-stationary phase interaction. In column chromatographic separations, it is desired to elute different components at different times so that fractions containing the pure component may be isolated. At the same time, it is undesirable for the components to interact too strongly with the column, or they will take too much time to elute. The balance between separation efficiency and separation time is a classical tradeoff to be made in chromatographic separations. A system that allows fine control over biomolecule/stationary phase interactions is highly advantageous.